Fig 2: Forskolin inhibits CCN1 via a monomeric actin-dependent mechanism.Cells were treated with 5 µg/ml latrunculin-B (A), 2 µM cytochalasin-D (B) or 0.5 µM jasplakinolide (C) for the indicated times; n = 3. CCN1 (?) and 36B4 (?) mRNA were quantified using qRT-PCT (A–C). Cells were infected with either Ad:Control (?) or Ad:Actin(R62D) (?) and CCN1 mRNA levels were quantified 24 h post infection; n = 5 (D). Cells were stimulated with 25 µM forskolin for 90 min either in the presence or absence of 0.5 µM jasplakinolide (E) or 2 µM cytochalasin-D (F) and CCN1 mRNA levels quantified; n = 4. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001 vs. Control.

Fig 3: Forskolin inhibits balloon-injury induced expression of CCN1 in rat carotid arteries in vivo.Immunohistochemical staining for CCN1 in balloon injured rat carotid arteries treated with forskolin. Immediately after balloon injury arteries were surrounded by 200 µl of 30% pluronic gel with or without forskolin. CCN1 (green) expression was analysed by immunohistochemistry in sections of either uninjured rat carotids or carotid arteries 7 days after balloon injury. Nuclei are stained blue with DAPI. Scale bar = 50 µm.

Fig 4: Inhibition of CCN1 expression by Adenosine, BAY60-6582 and Cicaprost.Cells were stimulated with 100 µM adenosine; n = 3 (A, D, E), 1 µg/ml BAY-60-6583; n = 3 (B, D, E) or 1 µM Cicaprost; n = 4 (C and F) for indicated times and analysed for CCN1 mRNA (A–C) and protein (D–F) expression qRT-PCR and Western blotting. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001. ? indicates control; ? indicates stimulated.

Fig 5: cAMP inhibits expression of CCN1 in VSMC.VSMCs were stimulated with for 2 h with the indicated concentrations of db-cAMP; n = 3 (A), with 500 µM db-cAMP for the indicated times; n = 3 (B) or with 25 µM forskolin for the indicated times; n = 3 (C). Quiescent VSMCs were stimulated with 50 ng/ml PDGFBB for 1 h with 25 µM forskolin as indicated; n = 6 (D). Total RNA was analysed for CCN1 (dashed line) and 36B4 mRNA (solid line) using RT-PCR (A–D). Cells were stimulated with 25 µM forskolin (E) or 500 µM db-cAMP (F) for indicated times and CCN1 protein quantified by Western blotting and densitometry at 8 h; n = 3; ? indicates control; ? indicates stimulated (G). Quiescent VSMCs were stimulated with 10% FCS (H) or 50 ng/ml PDGFBB (I) for indicated times and CCN1 protein quantified by Western blotting. (J) Quiescent VSMCs were stimulated with 10% FCS (H) or 50 ng/ml PDGFBB for 1 h in the presence of 25 µM forskolin or 500 µM db-cAMP, as indicated. CCN1 protein was quantified by Western blotting. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001.