Figure 7

Hoxc11-lacZ expression is restricted to a subset of smooth muscle cells in vessel explant culture. (A) Cellular outgrowth of explanted femoral artery/lateral marginal vein vessel segments (~2 mm; central dark ovoid structure in panel A) from young adult Hoxc11-lacZ transgenic mice [TG(Hoxc11/lacZ)62D9Awg] is comprised of β-gal-positive (blue) and -negative cells (black arrows in panel A, inset). (B) X-Gal labeling of cultured, femoral artery SMCs of Hoxc11-lacZ reporter mice reveals β-gal-positive (blue) and β-gal-negative cells as indicated by grey and white arrows, respectively. (B') Subsequent to X-Gal labeling, the cells shown in panel B were immunolabeled with antibodies specific for SMC marker Acta2 (formerly known as SMαA) as indicated by green fluorescence; note that both β-gal-positive and -negative cells as marked by grey and white arrows, respectively (grey and white arrows correspond to the same cells in panels B and B'), are labeled by anti-Acta2; nuclei were labeled with Hoechst 33342 (blue). (C/C'/C") Double-labeling of explant-derived, cultured femoral artery SMCs of TG(Hoxc11/lacZ)62D9Awg mice with antibodies against Hoxc11 (red signal, panel C) and anti-β-gal (green signal, panel C') results in a merged yellow signal in panel C". (D/E) Double-immunolabeling of cultured mitotic femoral artery SMCs with antibodies against Hoxc11 (red signal) and two standard SMC markers, including Transgelin (Tagln, formerly known as Smooth muscle protein 22-alpha; green signal, panel D), and Acta2 (green signal, panel E); note, overlap between Hoxc11 and Tagln signals in panel D results in yellow fluorescence, whereas the lesser degree of overlap between Hoxc11 and Acta2 signals in panel E results in discernible Hoxc11 (red) signal in the peri-nuclear region. Nuclei (blue) were labeled with Hoechst 33342.