Figure 6. Effect of cAMP elevating agents on miR-221/222 and p27 expression.

(A–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). (C–E) 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. (F) miR-221/222 regulation by mitogens, ERK, PGI₂, and cAMP.