FIG. 1.

CREB depletion with siRNA induces a dedifferentiated, proliferative, and synthetic phenotype in PA SMCs. Rat PA SMCs were transfected with nonspecific (Cntrl) or CREB-specific (CREB) double-stranded siRNAs as indicated and incubated overnight in DMEM containing 15% FCS. Cells were then transferred to DMEM containing 0.2% FCS for 72 h. (A) Whole-cell lysates and RNA were prepared from the SMCs and subjected to Western blotting (left panels) or semiquantitative reverse transcriptase PCR (right panels) for the factors indicated to the left of each pair of panels. (B) BrdU (10 μM) was added to the siRNA-transfected cells for 12 h. Cells were then fixed in PBS containing 4% paraformaldehyde and subjected to immunostaining for BrdU incorporation as an index of cell proliferation. Representative bright-field images are shown. Magnification, ×200. (C) Eight randomly selected microscope fields were counted for BrdU-positive and total cell numbers by a blinded observer. Data shown are average percentages of BrdU-positive cells. (D) siRNA-treated cells and untreated or PDGF-treated (25 ng/ml for 72 h) cells were subjected to pentachrome staining. Representative bright-field images are shown. Magnification, ×400.