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. 2010 Feb 4;151(4):1760–1772. doi: 10.1210/en.2009-0870

Figure 2.

Figure 2

eNOS activity is required for endothelial cell to promote actin cytoskeleton remodeling and cell plasticity in tanycytes. A, Schematic representations of the wild-type isoform of eNOS naturally expressed in endothelial cells of the median eminence and the DN-eNOS, which is a truncated form lacking the C terminus and catalytic activity. Whereas eNOS (600) antibodies recognize both the wild-type and truncated isoforms, eNOS(C-ter) antibodies recognize only the wild-type isoform of eNOS. B, Western blot analyses show truncated eNOS expression in ECMEs infected with the adenoviral vector driving DN-eNOS-expression. DN-eNOS is expressed in neither control conditions nor ECMEs infected solely with an adenoviral vector driving the expression of eGFP. Actin was used as a loading control. C, DN-eNOS is physically associated with wild-type eNOS in cells infected with the adenoviral vector driving the expression of the truncated eNOS. Protein extracts were immunoprecipitated (IP) with eNOS (C-ter) antibodies, electrophoresed to size fractionate immunoprecipitated species, and immunoblotted (IB) with eNOS (600) antibodies. D–G, Effects of eGFP (D and E) and DN-eNOS (F and G) expression in ECMEs on the morphology of cocultured tanycytes in the presence (E and G) or absence (D and F) of estradiol treatment (E2, 5 nm, 48 h). Whereas eGFP expression in ECMEs had no significant effect on endothelial cell-induced stress fiber formation (arrows) (D) or cytoplasmic retraction (long arrowheads) in tanycytes (E), DN-eNOS expression prevented endothelial cells from promoting such plastic changes in tanycytes cultured in the absence (F) or presence (G) of estradiol, respectively. Tanycytes cocultured with DN-eNOS-expressing ECMEs displayed cortical actin (arrowheads; F and G) as in the control conditions illustrated in Fig. 1B. Scale bar, 10 μm. H, Quantitative analysis of the plastic changes elicited by estradiol (E2) in tanycytes cocultured with ECMEs that do or do not express eGFP (Ad-eGFP) and DN-eNOS (Ad-DN-eNOS). Changes are measured by calculating the percentage of cells belonging to each phenotypic class.