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Blood, Vol. 113, Issue 22, 5669-5679, May 28, 2009
HDAC5 is a repressor of angiogenesis and determines the angiogenic gene expression pattern of endothelial cells
Blood Urbich et al.
113: 5669
Supplemental materials for: Urbich et al
Spheroid-based angiogenesis assay
Endothelial cell spheroids of defined cell number were generated as described previously.30 In brief, 14 hours after transfection HUVEC were suspended in culture medium containing 0.2% (wt/vol) carboxymethylcellulose (Sigma) and seeded in non-adherent round-bottom 96-well plates (Greiner, Frickenhausen, Germany). Under these conditions, all suspended cells contribute to the formation of a single spheroid per well of defined size and cell number (400 cells/spheroid). Spheroids were generated overnight, after which they were embedded into collagen gels. The spheroid containing gel was rapidly transferred into prewarmed 24-well plates and allowed to polymerize (30 minutes), then 100 µl endothelial basal medium with or without human FGF2 (30 ng/ml) was added on top of the gel. After 24 hours, pictures were taken using an Axiovert 100M microscope and a Plan-NEOFLUAR 10×/0.30 objective lens. In vitro capillary sprouting was quantified by measuring the cumulative sprout length per spheroid using AxioVision Rel 4.4 digital imaging software (Zeiss, Jena, Germany). To obtain a measure of the cumulative sprout length per spheroid, every sprout from 10 spheroids was assessed and from these data the mean cumulative sprout length per spheroid was calculated. In addition, the number of branch points and the number of sprouts was counted from 10 spheroids and calculated as mean number of branch points and mean number of sprouts per spheroid. Scratched wound assay
Migration of HUVEC was detected using a "scratched wound assay.31 Briefly, HUVEC were grown on 6 cm dishes previously labeled with a traced line. The cell monolayer was scraped with a sterile cell scraper to create a cell-free zone (width approximately 14 mm). Thereafter, cells were washed with medium. Endothelial cell migration was quantified by measuring the width of the cell-free zone (distance between the edges of the injured monolayer) at the time of injury and after 24 hours of cultivation using a computer-assisted microscope (Zeiss) at 5 distinct positions (every 5 mm).
Files in this Data Supplement:
- Table S1. siRNA oligonucleotides (PDF, 15.9 KB)
- Table S2. Oligonucleotides used for RT-PCR (PDF, 29 KB)
- Table S3. Oligonucleotides used for real-time PCR (PDF, 29.1 KB)
- Table S4. Oligonucleotides used for ChIP (PDF, 28.7 KB)
- Figure S1. Effect of siRNA transfection on viability (JPG, 328 KB)
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(A) HUVEC were transfected with siRNA oligonucleotides against HDAC5, HDAC7, and HDAC9. 24 hours after transfection, the effect on HDAC expression was assessed by quantitative real-time PCR. Data (delta Ct) are mean ± SEM, n = 3, * P<0.05 vs. control siRNA. (B/C) HUVEC were transfected with siRNA oligonucleotides against HDAC5, HDAC7, and HDAC9. 24 hours (B) and 48 hours (C) after transfection, the effect on viability of HUVEC was analyzed by MTT assay. Data are mean ± SEM, n = 4, * P<0.05 vs. control siRNA.
- Figure S2. HUVEC were transfected with scrambled (Scr) siRNA or HDAC5 siRNA (JPG, 243 KB)
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The cumulative length of sprouts, the number of branch points, and the number of sprouts per spheroid were determined in a spheroid-based sprouting assay. Data are mean ± SEM, n = 3, * P<0.05.
- Figure S3. Effect of HDAC5 siRNA transfection on HDAC5 protein expression (JPG, 82.9 KB)
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HUVEC were transfected with two different control siRNA oligonucleotides and two different siRNA oligonucleotides against HDAC5. After 48 hours, HDAC5 protein expression was analyzed by Western blot using an anti-HDAC5 antibody (Cell Signaling, 1:1000) and an anti-tubulin antibody as loading control. A representative Western Blot is shown.
- Figure S4. Effect of a second, unrelated siRNA oligonucleotide against HDAC5 on sprout length (% of scrambled siRNA; n=5) (JPG, 105 KB)
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- Figure S5. HUVEC were transfected with empty vector (mock), HDAC5 wt, or HDAC5 S259/498A (JPG, 320 KB)
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The cumulative length of sprouts, the number of branch points and the number of sprouts per spheroid was determined in a spheroid-based sprouting assay. Data are mean ± SEM, n = 4, * P<0.05 versus mock.
- Figure S6. HUVEC were transfected with empty vector (mock), HDAC5 wt, HDAC5 S259/498A, or PKD alone or in combination as indicated (JPG, 282 KB)
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(A) a representative Western blot is shown. (B) the cumulative length of sprouts per spheroid (% mock) was determined in a spheroid-based sprouting assay. Data are mean ± SEM, n = 4.
- Figure S7. Effect of HDAC5 siRNA on the suppression of HDAC5 compared to the other class IIa isoenzymes HDAC7 and HDAC9 (Affymetrix oligonucleotide array) (JPG, 86.1 KB)
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* P<0.05 vs. scrambled siRNA-transfected HUVEC (n=3).
- Figure S8. mRNA expression of selected genes involved in angiogenesis in endothelial cells (JPG, 348 KB)
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The mRNA expression of selected genes relevant to angiogenesis in HUVEC transfected with scrambled siRNA or HDAC5 siRNA after 24 h is summarized. Data are normalized mean fluorescence intensity ± SEM (n=3). In case of two or more different oligonucleotides representing one gene, two or more bars are shown.
- Figure S9. mRNA expression of selected genes involved in angiogenesis in endothelial cells (JPG, 319 KB)
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The mRNA expression of selected genes relevant to angiogenesis in HUVEC transfected with scrambled siRNA or HDAC5 siRNA after 24 h is summarized. Data are normalized mean fluorescence intensity ± SEM (n=3). In case of two or more different oligonucleotides representing one gene, two or more bars are shown. Please note that different oligonucleotides can represent different splice variants and soluble isoforms. * P<0.05 vs. scrambled.
- Figure S10. The ChIP was performed in HUVEC overexpressing myc-tagged HDAC5 wt or HDAC5 S259/498A mutant using a myc antibody (JPG, 118 KB)
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qPCR with primers detecting the indicated promoter regions of the FGF2 and Slit2 genes are shown. Data are mean ± SEM, n = 3, * P<0.05 vs. mock.
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