Clinical data: ages of patients at the time of resection and location of the lesions.

<p>Sample No.5 and No.6 of senile hemangioma were obtained from same patient.</p

The expression profiles of miRNAs as measured with the PCR array.

<p>A mixture of equal amounts of miRNAs from 3 senile hemangioma, 3 venous malformation or 3 angiosarcoma were prepared, and miRNA expression profile in each tumor in vivo was evaluated using RT² Profiler PCR Array. The raw threshold cycle (Ct) was normalized using the values of small RNA housekeeping genes. ΔΔCt (the raw Ct of each miRNA – Ct of small RNA housekeeping genes) were shown.</p

The expression of angiogenic factors or the structure of vessels.

<p>(a) Mean relative transcript levels of HIF-1α, VEGFR1 or VEGFR2 (normalized to GAPDH) in tissues from 7 normal skin (normal) or vascular anomalies such as 7 senile hemangioma (SH), 3 vascular malformation (VM), 4 angiosarcoma (AS), 4 venous lake (VL) and 3 infantile hemangioma (IH) by real-time quantitative PCR. The transcript levels in samples of normal skin were set at 1. Error bars represent SD of +1. (b) The expression of α-smooth muscle actin (SMA) or type IV collagen (COL4) in the affected vessels of senile hemangioma (SH), vascular malformation (VM) and venous lake (VL). Paraffin sections were subjected to immunohistochemical analysis with antibodies against α-SMA or COL4 as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014334#s2" target="_blank">Materials and Methods</a>’. Results are representative of several cases.</p

The immunoreactivity for mir-424 in senile hemangioma.

<p>In situ detection of mir-424 in paraffin-embedded, formalin-fixed tissues of normal skin (a) and senile hemangioma (SH, b). Nucleus was counterstained with nuclear fast red (magnification, ×200). The immunoreactivity for mir-424 (blue) is indicated by arrows. Results are representative of 3 normal skins and 3 SH.</p

The immunoreactivity for MEK1 and cyclin E1 in senile hemangioma.

<p>Increased expression of MEK1 (a) or cyclin E1 (b) in ECs of senile hemangioma. Sections of normal skin or vascular anomalies were stained with antibodies against MEK1 or cyclin E1 (green) and CD34 (red). SH; senile hemangioma, VM; vascular malformation.</p

The transcript levels of MEK1 and cyclin E1 in senile hemangioma.

<p>Mean relative transcript levels of MEK1 (white bars) and cyclin E1 (black bars) in the tissues from normal skin (normal) or vascular anomalies such as senile hemangioma (SH), vascular malformation (VM), angiosarcoma (AS), venous lake (VL) and infantile hemangioma (IH) by real-time quantitative PCR. The transcript levels in normal skin were set at 1.</p

The association of mir-424 with MEK1 or cyclin E1.

<p>(a, b) mir-424 inhibitor induces the protein expression of MEK1 and cyclin E1 but not mRNA expression in human dermal microvascular ECs. Cells were transfected with the control inhibitor or mir-424 inhibitor for 48 hours. (a) Relative amounts of transcripts (normalized with GAPDH) determined in total RNA with quantitative PCR. Error bars represent SD of +1. (b) Lysates from cells transfected with control or mir-424 inhibitor were subjected to immunoblotting with antibody for MEK1 or cyclin E1. The same membrane was then stripped and reprobed with anti β-actin antibody as a loading control. (c, d) mir-424/MEK1/cyclin E1 pathway altered the cell proliferation activity. (c) HDMECs at a density of 5×10³ cells/well in 24-well culture plates were transfected with control inhibitor or inhibitors specific for mir-424 or mir-1. After 48 hours, the number of cells was counted with a Colter® Particle Counter (Beckman Coulter, Fullerton, CA) as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014334#s2" target="_blank">Materials and Methods</a>’. The value in the cells transfected with control inhibitor were set at 1. The mean and SD from 3 separate experiments are shown. * P<0.05 in comparison to the value in the cells transfected with control inhibitor. (d) HDMECs at a density of 1×10⁴ cells/well in 24-well culture plates were transfected with control siRNA or siRNA specific for MEK1 or cyclin E1. After 72 hours, the cell number was counted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014334#pone-0014334-g007" target="_blank">Fig. 7c</a>.</p