Loss of NF1 expression is sufficient to induce endothelial cell proliferation.

<p>(A) pSIREN Control and <i>NF1</i> knockdown primary endothelial cells were grown in complete media under sub-confluent conditions and cumulative population doublings were recorded over the course of 25 days as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049222#pone.0049222-Bajaj1" target="_blank">[10]</a>. (B) Control or NF1 knockdown endothelial cells were serum deprived for 24 h prior to cells being pulsed with BrdU for 3 h. BrdU positive cells were quantified and the data was graphed as the % positive cells compared to total cell number. Data are presented as the mean with error bars representing the standard error (**p<0.01). (C) In parallel to the experiments performed in (B), serum starved cell lysates were made after 24 h and probed with antibodies against cyclin D1, p27, phospho p27, and ERK2. ERK2 is used to insure equal loading of lysate. (D) In three independent experiments similar to those performed in (C), results were quantified and data expressed as fold-change from control. Error bars represent standard error of the mean (*p<0.05).