Effect of cAMP elevating agents on miR-221/222 and p27 expression.

<p>(<b>A–B</b>) Quiescent early passage VSMCs from wild-type were stimulated with 10% FBS in the absence (control) or presence of 50 µM U0126 (U0), 1 mM 8Br-cAMP, or 100 µM Forskolin (Fsk). In A, total RNA was extracted at 24 h, and miR-221/222 expression levels were determined by RT-qPCR. Results show mean ± SE, n = 3−4. In B, total protein was extracted at 24 h and analyzed by western blotting for p27, dually phosphorylated ERK (pERK), total ERK and GAPDH (loading control). (<b>C–E</b>) The experiment in A was repeated with wild-type and p27-null VSMCs in 6-well dishes containing coverslips and EdU. In C, coverslips were fixed at 48 h and stained for EdU; results are plotted relative to the FBS-treated control; n = 3. In D-E, total RNA was extracted at 24 h, and miR-221 or miR-222 expression levels were determined by RT-qPCR. Results show mean ± SD, n = 2. (<b>F</b>) miR-221/222 regulation by mitogens, ERK, PGI₂, and cAMP.</p

miR-221/222 and Skp2 mRNA expression in injured femoral arteries.

<p>Male SMA-GFP (5–6 mo.) mice were subjected to fine-wire femoral artery injury. Injured regions of femoral arteries (as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056140#pone-0056140-g001" target="_blank">Fig. 1A</a>) and uninjured control femoral arteries were micro-dissected, and RNA was isolated. RT-qPCR was performed for miRNA221, miRNA222 and Skp2 expression. Gene expression for each mouse was expressed as fold increase (injured regions relative to uninjured control).</p

Transcript profiling reveals that miR-221/222 is induced after vascular injury in vivo.

<p>(<b>A</b>) Male SMA-GFP mice (5–6 mo) were subjected to fine-wire femoral artery injury. Injured arteries were isolated, carefully opened, and immediately imaged for GFP fluorescence. A representative image of an uninjured and injured femoral artery is shown for a single mouse. The bracket shows a region of vascular injury. (<b>B</b>) Uninjured femoral arteries and GFP-negative regions of injured femoral arteries were collected for transcript profiling. Genes differentially expressed in these tissues were plotted against the Gene Ontology (GO) category, Cellular Process. (<b>C</b>) Interaction map showing upstream regulators of p27 that are differentially expressed in injured vs. uninjured femoral arteries as determined by Ingenuity Pathway Analysis (IPA) of the microarray data. Green and red represent induction and repression, respectively. Upstream p27 regulators in the IPA database that were not differentially expressed during in vivo response to injury are uncolored. The boxed region of interest at the bottom of the interaction map is expanded below to highlight the induction of miR-221 (green oval). (<b>D</b>) Quiescent early passage mouse VSMCs were stimulated with 10% FBS for 24 h. Total RNA was collected, and the levels of miR-221/222 and Skp2 mRNA were determined by RT-qPCR. Results show mean ± SD, n = 2.</p

Effects of miR-221/222 and Skp2 on p27 levels during cell cycling and cell cycle inhibition.

<p>(<b>A</b>) Early passage VSMCs from wild-type or p27T187A mice were transiently transfected with control or anti-miR-222. Transfected cells were serum starved and stimulated with 10% FBS for 24 h before being collected and analyzed by western blotting for p27 and actin (loading control). (<b>B</b>) Cells were also serum starved and stimulated with 10% FBS for 48 h on cover slips; collected coverslips were used to determine EdU incorporation. Results show mean ± SD, n = 3.</p

miR-221/222 is a primary target of PGI₂.

<p>Quiescent early passage VSMCs from wild-type or p27-null mice were stimulated with 10% FBS in the absence (control; C) or presence of 200 nM cicaprost (cica). (<b>A</b>) Total RNA was extracted at 24 h, and Skp2 mRNA levels were determined by RT-qPCR. Results show mean ± SD, n = 2. (<b>B</b>) Total protein was extracted and analyzed by western blotting for p27, Skp2 and actin (loading control). <b>(C)</b> Total RNA was extracted at 24 h, and miR-221/222 levels were determined by RT-qPCR. Results show mean ± SD, n = 2.</p

Regulation of G1 and S phase p27 by PGI₂.

<p>(<b>A–B</b>) Quiescent early passage explant VSMCs from wild-type or p27T187A mice were stimulated with 10% FBS for 24 or 30 h in the absence or presence of 200 nM cicaprost. Total protein was extracted at the indicated times and analyzed by western blotting for p27 and actin (loading control). The percent EdU incorporation, determined from coverslips included in the experiments, is shown in italics. (<b>C–D</b>) Quiescent VSMCs were treated with 10 µM MG132 or DMSO (vehicle control) and stimulated with 10% FBS for the indicated times. Total protein was extracted and analyzed for p27 protein levels by western blotting and actin (loading control). Total RNA was extracted at 9 (n = 3) and 18 h (n = 2) and analyzed for miR221/222 by RT-qPCR. RT-qPCR results show mean ± SD. Note that G1 phase downregulation of p27 was evident 9 h after FBS stimulation in this experiment, affording us more time points to document the inhibitory effect of MG132 on G1 phase miR221/222.</p

miR221/222 regulation by PGI2 and role in mitogensis.

<p>(<b>A</b>) Serum-starved VMSCs from wild-type mice were incubated with 10% FBS and 2 nM cicaprost for selected times in the presence of EdU. Top Results show mean ± SD, n = 2. Bottom panel: S phase entry was assessed and plotted as percent maximal EdU incorporation. Results show mean ± SD, n = 3. (<b>B–C</b>) Early passage wild-type VSMCs were transiently transfected with an expression plasmid for pCDNA (control) or miRNA-222. The cells were serum starved and stimulated with 10% FBS for 24 (panel B) or 48 (panel C) h in the absence or presence of 200 nM cicaprost (cica) before being collected and analyzed for p27 levels by western blotting or S phase entry by EdU incorporation. Results in panel C show mean ± SD, n = 3.</p