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Blood, Vol. 108, Issue 9, 3068-3071, November 1, 2006
MicroRNAs modulate the angiogenic properties of HUVECs
Blood Poliseno et al.
108: 3068
Supplemental materials for: Poliseno et al
Files in this Data Supplement:
- Supplemental Methods (PDF, 108 KB)
- Figure S1. c-Kit and pri-miR-221/2 expression in HUVEC (PDF, 62.4 KB) -
A c-Kit expression. c-Kit expression was detected by: immunohistochemistry (upper), HUVEC incubated with both the primary and secondary antibody (right) show a staining that is absent when they were incubated only with the secondary antibody (left). Immunofluorescence (lower). Dark area: distribution of fluorescence relative to PBS incubation (background); light area: distribution of fluorescence relative to primary and secondary antibodies incubation. A representative experiment of three performed is reported.
B. pri-miR-221 and pri-miR-222 expression. (upper) Schematic representation of the genomic organization of miR-221 and miR-222 genes on X chromosome. Grey box: pre-miRNA; black box: mature miRNA. (lower) pri-miR-221 (left) was amplified by RT-PCR using 1F and 1R primers, pri-miR-222 (middle) using 2F and 2R primers. The presence of a precursor containing both of them (right) resulted from the amplification with 2F and 1R primers. A representative experiment of three performed is reported. L1: 100bp ladder; L2: 1kb ladder; 1: -RT; 2: +RT; 3: genomic DNA.
- Figure S2. EGFP reporter assay (PDF, 30 KB) -
A. Schematic representation of c-Kit 3′UTR. The position of predicted miRNA target sites along the 3′UTR of c-kit is indicated with black boxes. 215 target site is predicted by miRanda for miR-221, while 1030 and 1959 target sites are predicted by TargetScan for both miR-221 and miR-222. 3′UTR nucleotides are counted starting from the first nucleotide after the STOP codon. Numbers refer to the most upstream base of the seed match.
B. Determination of 1959 target site expression in HUVEC. (upper) The presence of 1959 target site within mature c-kit mRNA was verified by RT-PCR using spanning f2 and r2 primers, that ampify a 522bp fragment. L1: 100bp ladder; g: genomic DNA. (lower) Sequencing of the obtained PCR fragment. 1959 target site is in bold.
C. Schematic representation of hybrid EGFP reporter plasmids. The sequence of each single predicted target site (215, 1030 and 1959, grey box) or the whole 3′UTR sequence of c-kit (dotted box) were cloned downstream of EGFP ORF (white box) within the Multi Cloning Site of pEGFP-C1 plasmid (light grey box). p215, p1030, p1959 and p3′UTR plasmids were in this way obtained. An in frame STOP codon is inserted between the EGFP ORF and cloned c-kit fragment. Black arrow: CMV promoter.
D. Schematic representation of miRNA expression plasmids. Each ∼500bp pri-miRNA sequence (pri-miR-221, pri-miR-222 and pri-miR-26a, white box) was cloned downstream of CMV promoter (black arrow) within the Multi Cloning Site of pCMV-MCS plasmid (grey box).
E. Relative activity of p221 and p222 on hybrid reporter plasmids. p221 (grey) and p222 (white) expression plasmids were challenged for their ability to decrease the fluorescence of p215, p1030, p1959 and p3∼UTR hybrid EGFP reporter plasmids. Relative activity was calculated as 1-(relative residual fluorescence). Relative residual fluorescence was calculated as the ratio between the residual fluorescence of cells transfected with p221 or p222 and the residual fluorescence of cell transfected with p26a.
- Figure S3. HUVEC purity assessment (PDF, 31.4 KB) -
FACS analysis of HUVEC incubated with: anti-CD31 primary antibody; E1/1 primary antibody, which recognizes a constitutive and non—cytokine-inducible endothelial cell antigen; E selectin primary antibody. E selectin was detected after 6h exposure of HUVEC to 1µg/ml LPS. Dark area: distribution of fluorescence relative to secondary antibody incubation (background); light area: distribution of fluorescence relative to primary and secondary antibodies incubation. A representative experiment of three performed is reported for each purity assessment.
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