Volume 20, Issue 1, Pages 45-52 (October 2005) Downregulated REST Transcription Factor Is a Switch Enabling Critical Potassium Channel Expression and Cell Proliferation  Alex Cheong, Andrew J. Bingham, Jing Li, Bhaskar Kumar, Piruthivi Sukumar, Christopher Munsch, Noel J. Buckley, Craig B. Neylon, Karen E. Porter, David J. Beech, Ian C. Wood  Molecular Cell  Volume 20, Issue 1, Pages 45-52 (October 2005) DOI: 10.1016/j.molcel.2005.08.030 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 Identification of Functional RE1 Sites in KCNN4 Genes (A) Sequence alignment of regions of human, mouse, and rat KCNN4. The conserved RE1 sequences that align to the RE1 consensus NTYAGMRCCNNRGMSAG (N = A, C, G, or T; Y = C or T; M = A or C; R = A or G; S = C or G) (Bruce et al., 2004) are boxed and labeled Site A and Site B. (B) Gel mobility shift assay using nuclear extracts from cells that have well-characterized high expression of REST (JTC-19 cells; [Belyaev et al., 2004; Wood et al., 2003]) and radiolabeled SCN2A2 probe (Wood et al., 2003). Abbreviations: none, no competitor; RE1, RE1 competitor (for [A] or [B], see Figure 1A); and Sp1, Sp1 competitor. The specific REST-DNA complex is identified by an arrow, and each lane is labeled with the oligonucleotides that were used for competition. The result is representative of three independent experiments. (C) Representation of KCNN4 constructs containing wild-type (wt) or RE1-mutated promoter regions driving expression of the luciferase gene. (D) Luciferase activity driven from wt or mutant KCNN4 promoter transfected into HEK cells; expression, normalized for transfection efficiency, is expressed relative to wt promoter (mean ± SEM, n = 4). Molecular Cell 2005 20, 45-52DOI: (10.1016/j.molcel.2005.08.030) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 REST Is Expressed in Vascular Smooth Muscle and Recruited to KCNN4 Gene In Situ (A) RT-PCR on mRNA isolated from endothelium- and adventitia-denuded mouse aorta by using REST-specific primers. PCR products from mRNA treated with (lane 1) and without (lane 2) reverse transcriptase (RT) were electrophoresed through agarose gel. Abbreviation: M, 25 bp ladder. (B) Melt curve analysis of real-time PCR product indicating a single specific product with RT (+RT) and absence of product without RT (no RT). Abbreviations: F, fluorescence; t, temperature. (C) Western blot of protein extracted from endothelium- and adventitia-denuded mouse aorta by using anti-REST antibody. Molecular weights of markers are shown on the left; REST protein is indicated. (D–F) Fresh human saphenous vein stained with haematoxylin (pink) and eosin (purple) (D). Scale bar, 500 μm. (E–F) Immunohistochemistry showing positive REST staining (brown). The medial layer is shown for labeling with anti-REST (E) or preimmune (F) serum. Scale bar, 50 μm. (G) Electro mobility shift assay using the SCN2A2 probe with nuclear extracts obtained from human saphenous vein smooth muscle. Abbreviations: none, no competitor; RE1, RE1 competitor; Sp1, Sp1 competitor; and REST Ab, anti REST antibody. The positions of the REST-DNA and REST-DNA-Ab complexes are indicated by arrows. (H) Quantification of KCNN4 RE1 and M4 non-RE1 (M4 coding) sequences precipitated by anti-REST antibody in freshly isolated medial layer of saphenous vein (mean ± SEM, n = 6 patients). Dotted line indicates the level of background precipitation. Molecular Cell 2005 20, 45-52DOI: (10.1016/j.molcel.2005.08.030) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Changes in REST and KCa3.1 in Proliferating Cells (A and B) Freshly isolated (A) and cultured (B) mouse aorta smooth muscle cells stained with antibody to smooth muscle α-actin (red). DAPI counterstain (blue) indicates nuclei. (C) Real-time RT-PCR quantification of REST mRNA levels in freshly isolated and proliferating mouse aorta smooth muscle cells (mean ± SEM, n = 7 for fresh and 6 for proliferating, *p < 0.05). Expression levels are normalized relative to 16S mitochondrial RNA, which was not significantly different between fresh and proliferating cells. (D) As for (C) except KCNN4 expression (mean ± SEM, n = 3 for each, *p < 0.05). (E and F) Immunohistochemistry of a saphenous vein from one patient, showing the vein before (E) and 2 weeks after organ culture (F). The stain is of smooth muscle α-actin (orange). Fresh neointimal growth, comprising smooth muscle cells, is indicated by arrows. Scale bar, 1 mm. (G and H) Immunohistochemistry of 2 week organ-cultured human saphenous vein with anti-REST serum. Positive staining is brown, the nuclear counter stain is purple. Arrowheads in (G) indicate REST-positive nuclei in medial layer and in (H) indicate REST-negative nuclei in neo-intima. Scale bar, 5 μm. (I) Immunohistochemistry of cultured vein with anti-KCa3.1 serum showing positive staining (brown) confined to the neo-intimal smooth muscle cells. Scale bar, 50 μm. Staining was absent when the primary antibody was omitted (data not shown). Molecular Cell 2005 20, 45-52DOI: (10.1016/j.molcel.2005.08.030) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 Functional Analyses of KCa3.1 in Human Saphenous Vein (A and B) Example sections of saphenous vein from the same patient 2 weeks after organ culture in vehicle control ([A], DMSO) or 200 nM TRAM-34 (B). The images show autofluorescence, which facilitates delineation of the neointimal boundaries (indicated by dotted lines). The arrow indicates the neointimal thickness, and the scale bar is 0.5 mm. (C) Mean ± SEM area of the neointima after TRAM-34 treatment and normalized to its respective control (n = 7 patients, *p = 0.03). (D–F) Fluorescence (F) from X-rhod-1 loaded into cultured human saphenous vein smooth muscle cells. (D and E) Comparison of mean ± SEM data (×103, n = 12) for cells from the same patient but cultured in the presence of 10% serum (D) or 0.4% serum (E). The experiments show calcium-entry signals in response to addition of 0.2 mM extracellular calcium in the presence or absence of 200 nM TRAM-34. DMSO was the solvent and was thus included as a control. (F) is as for (D) and (E) but showing the mean ± SEM effect of 200 nM TRAM-34 in cells infected with Adenovirus expressing full-length REST (Ad-FL, **p = 0.007, n = 8) or dominant-negative mutant REST (Ad-DN, **p = 0.001, n = 4) normalized to parallel Adenoviral vector controls (Ad-Ctrl). Molecular Cell 2005 20, 45-52DOI: (10.1016/j.molcel.2005.08.030) Copyright © 2005 Elsevier Inc. Terms and Conditions