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Fig. 6. Overexpression of Hand1 and Hand2 in primary ventricular cultures. (A) Anti-ß-galactosidase immunocytochemistry of right ventricular transgenic cardiomyoytes after transfection with a CMV-Hand1 expression vector showing a ß-galactosidase-positive nucleus (white arrowhead). (B) Co-immunocytochemistry with an anti-myc tag antibody showing three positive nuclei (green arrowheads). (C) Hoechst staining reveals all nuclei in the field. (D) Overexpression of Hand1 and Hand2 under the control of a CMV promoter following adenoviral infection of primary cardiomyocytes prepared from left and right free ventricular walls of Mlc3f-nlacZ-2 hearts. Cells were stained with X-gal (dark nuclei) and photographed under a fluorescent filter. All adenoviral-infected cells express green fluorescent protein. Despite a high degree of infection, Mlc3f-nlacZ-2 transgene expression status is unaffected by overexpression of either Hand gene or of CMV-GFP alone. Control shows mock-infected X-gal-stained cells. White arrowheads indicate right ventricular cells infected with a Hand1 encoding adenovirus that do not express the transgene and left ventricular cells infected with a Hand2 encoding adenovirus that continue to express the transgene. (E) Overexpression of Tbx5 under the control of a CMV promoter following adenoviral infection of right or left ventricular cardiomyocte cultures does not modify Mlc3f-nlacZ-2 expression status in the presence or absence of trichostatin A (TSA), and co-infection with a Hand1 encoding adenovirus. Cells were stained with X-gal (dark nuclei). Scale bars: 25 µm.
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